1. Field of the Invention
The present invention relates to a method of making microtiter plates, which each have an array of microstructures, comprising at least microcups. The invention also relates to a microtiter plate.
2. Description of the Related Art
Microtiter plates, which are known from numerous disclosures, e.g. DE 197 36 630 A1 or DE 197 40 806 C2, are plate-shaped base bodies provided with a plurality of very small reaction chambers. The reaction chambers, also called cavities or cups, are arranged in rows and columns to form a multi-cellular or honeycomb structure. The smallest amount of a liquid sample, for example of blood for diagnosis of medicinal parameters or diseases detectable in blood or water for monitoring water quality, are put in each reaction chamber. During the testing a chemical or biological reaction or the like takes place, which is accompanied by a coloration or discoloration of the liquid samples. The color change resulting from the reaction is usually monitored optically or electro-optically. For this latter reason the microtiter plates are constructed from a transparent material.
The word part “titer” in the words “microtiter plate” originates or is derived from titration analysis, in which it is defined as the content of a dissolved reagent in a standard solution.
The micro titer plates are provided with cavities of different diameters, for example in a range of from 3 to 7 mm. Typically they have dimensions of 120 mm×80 mm.
It is known to make microtiter plates composed of glass or transparent plastic. Microtiter plates composed of glass have little self-fluorescence as well as high chemical resistance and thus a great service life. They are very difficult to manufacture with conservative methods and thus quite expensive. Significant breakage danger and thus only very limited design possibilities exist for microtiter plates made of glass, as is the case with all glass products. Furthermore the tolerances attainable with glass, which depend on material and process conditions, are very much poorer than with typical injection molded plastic materials.
Micro titer plates composed of plastic are of course easy to make, e.g. by micro-injection molding. However they have very limited chemical resistance, especially to organic solvents, and little heat resistance. Furthermore the transparency of the cavity bottoms is limited to short wavelength light, so that optical determinations of the results of treatment or reaction are limited to only or dependent on UV light.
Micro titer plates have a special importance in biotechnology. Since the start of the nineties micro reaction systems for biotechnology, in which processes, such as measuring, mixing, synthesizing and analyzing of biochemical reactions are performed, have been investigated. The efficient and flexible employment of these systems in comparison to traditional macroscopic laboratory components and structures is significant, especially in the case of the micro titer plate. Biotechnology in recent years is thus one of the fastest growing and most innovative application areas for micro technology.
Microtiter plates like these micro reaction systems are used for rapid parallel high throughput tests (abbreviated HTS) for analysis of biochemical reactions by means of interferometry and fluorescence spectroscopy. Known microtiter plates of this type typically have 96 1.536 microcups with volumes in the pl to μl range on a total microtiter plate surface area of less than 100 cm2. The material cost factor in analyses of pharmaceutical, bio- and genetic engineering products can be significantly reduced because of the small reaction volumes. Also the large number of parallel tests leads to rapid and economical analysis. The n×m microcups are arranged in arrays with grid numbers of (n′×8)×(m′×12). The grid width between the microcups amounts to up to a few millimeters. The individual microcups can be uniformly and simultaneously filled with reaction participants by means of capillary action through micro channels. The micro channels have channel widths and depths greater than 50 μm. The microcups are filled in parallel with other reaction partners. This parallel filling occurs by means of transfer systems, which comprise a base plate with an appropriate number of rods. The rods are dipped in the reagents. A definite amount of material adheres to the rod tips by adhesion. The transfer of material occurs by dipping the rod tips with the material adhering on them in the microcups. An additional method for filling the microcups makes use of the principle of ink-jet printers instead of the rods.
The material selected for making the micro reaction systems depends particularly on the chemical and thermal material properties. The importance of borosilicate glass as the standard material for making macroscopic systems is carried over in micro reaction technology. Besides high chemical and heat resistance in comparison to most plastics, which lead to a long service life for the micro titer plates, these glasses have a higher degree of transmittance in the UV, VIS and IR spectral ranges in comparison to metals and ceramics. This is an important prerequisite for spectral analysis of chemical reactions. Glass characterized by very little self-fluorescence is suitable for fluorescence spectroscopy analyses, which are often performed in micro titer plates. Borosilicate glass acting as an insulating material allows the future automation of processes in a micro titer plate by combination of micro reaction structures with microelectronic elements in the glass substrate.
Photolithography combined with etching is the dominant process used for making the foregoing microtiter plates from glass. Photolithography is based on the preparation of a contour mask with detailed microstructure. The blank for the contour mask comprises a quartz glass substrate with a chromium absorber layer and an overlying radiation-resistant photo resist layer. The photo resist is partially irradiated with electron, ion or laser radiation according to the desired detailed microstructure and subsequently developed. The processing time for the irradiation of the photo resist can amount to several hours according to the mask size and required resolution. When a positive resist is used the irradiated regions are subsequently dissolved chemically from the surface. When a negative resist is used the regions which are not irradiated are dissolved chemically from the surface. During subsequent etching the photo resist portions remaining on the surface act as an etching stop, whereby the scaled microstructure is produced in the Cr-absorber layer. By removing (“stripping”)the remaining photo resist the contour mask required for the microstructuring is produced. After that the structure on the contour mask is transferred on the substrate covered with photo lacquer. This process corresponds to the irradiation during the manufacture of the contour mask, in which the irradiation times are considerably reduced in comparison to the mask manufacture.
Material is removed from the glass substrate by means of wet chemical, RIE- or dry etching methods. HF or an HF/H2SO4 acid mixture is used for wet chemical etching, while RIE- and dry etching are performed exclusively with fluorine compounds. During isotropic wet chemical etching the ingredients of the glass network are converted into fluorides by HF or into sulfates by H2SO4 at temperatures of 15° C.<T<70° C. and dissolved uniformly from the network. Large aspect ratios are not possible because of the isotropy of the wet chemical etching and because of under etching under the photo lacquer. Anisotropic etching can be achieved in RIE and dry etching methods by using passivating layers for protection of the not removed substrate regions and by preferred directions in irradiation dependent excitation of the etching processes. The etching processes can make exactly shaped microstructures with surface roughness Ra<10 nm. The structure depth for microchannels with nearly perpendicular edges amounts to <25 μm. However when the etching speed or rate is large the mask/substrate selectivity of the etching and the exactness of the formed structures deteriorate so that the RIE etching rate for silicate glass is usually in range from 50 to 100 nm/min.
These processes are usually very expensive because of the large number of treatment steps.
A laser irradiation process, which is especially suited for prototype, small-scale and medium-scale production of microtiter plates, is an additional known method for making microtiter plates. Structures are produced in the in-range with material removal rates RAbl=150 nm/pulse with the VUV laser radiation (wavelength λL=157 nm) of an F2 excimer laser. The proportion of photochemical material removal is larger than with long-wavelength laser radiation because of the large photon energy EPh=7.9 ev. This clearly reduces the melt deposition at the structured edges. The wavelength and the nonuniform local power density distribution of the laser radiation for that require considerable effort for guiding and forming the beam in a vacuum system. The manufacture of optics with sufficient service life is also a problem.
The small pulse energy of the F2 laser beam, EP=60 mJ, in comparison to ArF and KrF excimer laser beams permits small surface area static mask projection methods. Exactly formed microstructures are made with UV excimer laser beams with wavelengths of λL=193 nm (ArF), 248 (KrF) and 308 nm (XeCl) depending on the processed glass material. The removal rates RAbl are in a size range between RAbl>50 nm/pulse (λL=193 nm) and RAbl<6 nm/pulse (λL=308 nm). Lateral structure dimensions of a several tens of micrometers are achieved. Especially with microstructuring with laser radiation of wavelength λL=193 nm exact microstructures are formed in borosilicate glass, while at larger excimer laser wavelengths with pulse durations in the ns range the structural precision is influenced by fissures and conchoidal fractures in the glass. Micro holes with aspect ratios>1:1 and diameters>200 μm are made with ArF and KrF excimer laser radiation in different silicate glasses. Material is removed by laser beams in the visible wavelength range, e.g. copper vapor laser beams (λL=511 nm and λL=578 nm) and dye laser beams (λL=615 nm), with large pulse peak power densities with ultra short pulse duration or with large average pulse power with large repetition rates. In the visible range particularly pulse duration in the femto second and pico second range and a power density in a range of pL>1012 W/cm2 are used. The probability of two-photon absorption is increased at this power density of the femto second and pico second pulse. The interaction of the laser beam with the expanding plasma is decreased with the femto second and pico second pulse. The resulting large photochemical material removal leads to less melt release with removal rates of RAbl=400 nm/pulse in sodium-potassium silicate glass. Laser beams with pulse duration in the nanosecond range and repetition rates>1 kHz in quartz glass and borosilicate glass are used to make micro holes with aspect ratios<50:1 and diameters of greater than 200 μm.
Sources for laser beams with ultra short pulse duration are complex to handle and commercially available sources for these laser beams are limited in their availability. Material removal by laser beams in the IR range was tested with Nd:YAG laser beams (λL=1.064 nm) and CO2 laser beams (λL=10.6 λm). Q-switched CO2 lasers are used e.g. for marking glass surfaces. The removal rates of the photochemical material removal with a CO2 laser beam reach values of RAbl=2 to 3 μm2/pulse. The achievable structural accuracy is however not sufficient for making microtiter plates, since considerable removal of melt occurs at the structure edges and fissures and fractures arise because of the photochemical material removal.
The method using laser beam removal is thus unsuitable for solution of the problem of economical large-scale manufacture of microtiter plates.
Conventional metal-working manufacturing methods, such as ultraprecision turning or milling, rapidly approach performance limits in regard to making complex microstructuros, such as microtiter plates.